Many cell products have been identified via analysis of their effect on targeted cell populations and subpopulations. Such products include a plethora of materials including growth factors, cytokines, and so forth. These substances are extremely powerful and are produced in vanishingly small amounts by normal cells.
One of the major goals of contemporary molecular biology is the identification and cloning of genes which produce the various materials generally discussed supra. While such genes have been identified for materials such as erythropoietin (see, e.g., U.S. Pat. No. 4,703,008), and interleukin-7 (see, e.g., U.S. Pat. No. 4,965,195), the relevant genetic material is not always available, or is not available as quickly as the art would like. In such cases, an important means of securing sufficient amounts of the material of interest is via the identification and isolation of cell lines which produce the desired product.
Examples of proteinaceous cell products which were first identified via cell lines which produced them are "granulocyte colony stimulating factor" or "G-CSF" (see U.S. Pat. No. 4,833,127), and interleukin-3 (see U.S. Pat. No. 4,658,018). The latter patent is of particular interest because, while the factor could not be completely characterized in terms of parameters such as molecular weight, isoelectric point, etc., its functional properties were clearly defined. Another example of this approach to factors of biological pertinence and their isolation from cell lines may be seen in Golde et al., U.S. Pat. No. 4,438,032. Of course, the artisan is also aware of the vast number of known monoclonal antibodies which are produced by hybridoma cell lines. Frequently, the most pertinent feature of these proteins is their specificity, and this is all that is required to identify them.
Although many factors involved in the formulation of cell growth have been identified, the diversity of different cell types is evidence of the need to continue work toward identifying and characterizing factors which have specific targets, i.e., which stimulate particular cell types.
The different cell types of a mature organism do not appear "sua sponte", rather, they develop from precursor cells which have the ability to self renew and to differentiate into different types of mature cells. These precursor cells are referred to as "stem cells" generally, and are an important resource to the field for studies on cellular development. The importance of stem cells to basic research can be seen in U.S. Pat. No. 5,061,620, to Tsukamoto et al., which involves the isolation and maintenance of hematopoietic stem cells. Various media, including IMDM, and RPMI are described as growth media which can be used to maintain these stem cells.
The stem cells which develop into colonic mucosa are also referred to as colonic crypt cells or as a population thereof. While it has been possible to isolate these cells from mixed culture, their cultivation has been difficult. See in this regard Whitehead et al., In Vitro Cellular and Developmental Biology 23(6): 436-442 (1987), hereinafter referred to as "In Vitro", the disclosure of which is incorporated by reference. The methodology described therein requires, inter alia, the use of collagen gels and feeder layers of bovine aortic cells for extended culture of the crypt cells. This methodology is involved, and not completely satisfactory for the cultivation of colonic crypt cells.
Tumor cells are frequently the source of cell lines which are high producers of stimulatory factors for their normal counterparts. With reference to the patent literature cited supra, e.g., the cell lines identified as producers of G-CSF and IL-3, e.g., were isolated from tumorous material.
Cell lines derived from colon tumors and which can be traced to colonic crypt cells are known, as per, e.g., Whitehead et al., JNCI 74(4): 759-762 (1985), the disclosure of which is incorporated by reference and which described cell line LIM 1215, whose origin is traced to colon crypt cells. Not all cell lines produce pertinent factors, however, and there is no guarantee or pattern as to which cell lines will produce a stimulatory factor.
Previous investigations yielded a cell line which produces a membrane associated factor not otherwise recognized by the art, and which acts as a mitogen on colonic crypt cells. The factor acts as a mitogen in that it stimulates DNA synthesis and proliferation of the targeted cells (i.e., colon crypt cells). The factor is secreted by the producing cell line into the growth medium, to yield a conditioned medium which stimulates proliferation of colonic crypt cells. It is this mitogen, the cell line producing it, and applications thereof, which were the subject of the parent and grandparent applications of the parent case. These two applications disclosed, in FIG. 5, and in example 8, the use of crude pituitary material "A2". This material is an extract of bovine pituitary glands, and has been shown to be a potent clonogen for colonic carcinoma cell line LIM 1215. See, e.g., Whitehead et al., J. Nat. Canc. Ins. 74:759-765 (1985). The fraction, as indicated, is crude; however, pituitary extracts have been stuided further and it has been found, surprisingly, that the peptide consisting of the amino acid sequence of SEQ ID NO: 1 is a powerful stimulator of colonic carcinoma cell proliferation. It is this aspect of the peptide which is the basis for the invention described herein.